Detection and localization of Rickettsia sp in mealybug
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Published on: February 4, 2018
Shalini Thakur Singh1, Jitendra Kumar, Asha Thomas
1Department of Zoology, Gut Biology Laboratory, University of Delhi, Delhi, India.
Abstract:
Mealybug, Phenacoccus solenopsis Tinsley, is a sap-sucking hemipteran insect. It is an agricultural pest that is now widely distributed in India. In this study we report the presence of Rickettsia from P. solenopsis. We constructed a 16S rRNA gene library to study the bacterial diversity associated with this insect and we found that all the clones from the library were only of Candidatus Tremblaya phenacola. This study also highlights that the normal protocol adopted to study the bacterial diversity from environmental sample, by preparation of a 16S rRNA gene library, does not work when the bacterial population is highly skewed in favor of one bacteria (primary endosymbiont in this case). Hence, we used bacterial genus specific polymerase chain reaction primers to test the presence of any of the widely known secondary endosymbionts associated with insects. We tested for the presence of Cardinium, Rickettsia, Wolbachia, and Arsenophonus in P. solenopsis collected from 10 different locations across India. Only Rickettsia was detected from four locations while we were not able to find any other bacteria. We confirmed the presence of these bacteria by localizing Rickettsia and the primary endosmbiont, Candidatus Tremblaya sp. to the bacteriocyte of P. solenopsis using fluorescent in situ hybridization.
Insights
Rickettsia bacteria were detected in the agricultural pest Phenacoccus solenopsis mealybug across India. Standard bacterial diversity methods failed, necessitating targeted PCR for detection of this significant Rickettsia endosymbiont.
Area of Science:
- Entomology
- Microbiology
- Genetics
Background:
- Phenacoccus solenopsis is a significant agricultural pest in India.
- Insect endosymbionts play crucial roles in host physiology and evolution.
- Previous studies on P. solenopsis bacterial communities were limited.
Purpose of the Study:
- To investigate the bacterial diversity within Phenacoccus solenopsis populations in India.
- To identify potential secondary bacterial endosymbionts in P. solenopsis.
- To evaluate standard bacterial diversity assessment methods for skewed microbial populations.
Main Methods:
- Construction and sequencing of a 16S rRNA gene library.
- Development and application of genus-specific PCR primers for insect endosymbionts (Cardinium, Rickettsia, Wolbachia, Arsenophonus).
- Fluorescent in situ hybridization (FISH) to localize bacteria within host bacteriocytes.
Main Results:
- The 16S rRNA gene library was dominated by Candidatus Tremblaya phenacola, obscuring other bacterial presence.
- Genus-specific PCR successfully detected Rickettsia in P. solenopsis from four of ten surveyed Indian locations.
- FISH confirmed the co-localization of Rickettsia and Candidatus Tremblaya sp. within the bacteriocytes of P. solenopsis.
Conclusions:
- Standard 16S rRNA gene library methods are insufficient for detecting low-abundance bacteria in hosts with a dominant primary endosymbiont.
- Rickettsia represents a common secondary endosymbiont in Phenacoccus solenopsis populations across India.
- Targeted PCR and FISH are effective methods for characterizing specific endosymbiont communities in insects.
